Experimental Methods for Protein Structure Determination

True (A)

An NMR structure calculated with ≥20 restraints per residue is equivalent to a 2-2.5 Å crystal structure.

<p>quality</p> Signup and view all the answers

<p>Restraints per residue = &gt; 18 Backbone rmsd = &lt; 0.3 Heavy-atom rmsd = &lt; 0.75 Ramachandran Plot quality (%) = &gt; 95</p> Signup and view all the answers

<p>&lt; 0.75 Å (C)</p> Signup and view all the answers

<p>False (B)</p> Signup and view all the answers

<p>Unusual bond lengths or bond angles</p> Signup and view all the answers

<p>Ribbon model (B)</p> Signup and view all the answers

<p>False (B)</p> Signup and view all the answers

<p>0.15 to 0.20</p> Signup and view all the answers

<p>non-crystalline</p> Signup and view all the answers

<p>R-factor = Measure of agreement between model and data Rfree = Global measure calculated from random reflections RMSD = Indicates deviation from stereochemical standards Ramachandran plot = Percentage of residues with unusual φ/ψ angles</p> Signup and view all the answers

<p>Ball-and-stick model (B)</p> Signup and view all the answers

<p>True (A)</p> Signup and view all the answers

<p>Errors in the model</p> Signup and view all the answers

<p>van der Waals</p> Signup and view all the answers

<p>To reduce protein solubility and create a supersaturated state (B)</p> Signup and view all the answers

<p>Cryo-EM (A)</p> Signup and view all the answers

<p>False (B)</p> Signup and view all the answers

<p>Hanging-drop method</p> Signup and view all the answers

<p>BC, CD</p> Signup and view all the answers

<p>Resulting spots on the detector (B)</p> Signup and view all the answers

<p>Vapor diffusion = Method for forming crystals by balancing concentrations X-ray diffraction = Technique used to analyze crystal structure Elastic scattering = Process where scattered waves maintain energy and wavelength Unit cell = Smallest repeating unit of a crystal structure</p> Signup and view all the answers

<p>False (B)</p> Signup and view all the answers

<p>It increases as water is lost from the drop.</p> Signup and view all the answers

<p>Different chemical environments (C)</p> Signup and view all the answers

<p>False (B)</p> Signup and view all the answers

<p>Dipole-dipole relaxation between protons in close proximity</p> Signup and view all the answers

<p>Conformational dynamics or lack of data (A)</p> Signup and view all the answers

<p>COSY</p> Signup and view all the answers

<p>False (B)</p> Signup and view all the answers

<p>COSY = Detects neighbors up to three bonds apart TOCSY = Detects entire amino acid spin systems NOESY = Measures spatial proximity of protons Scalar coupling = Coupling through covalent bonds</p> Signup and view all the answers

<p>Nuclear Overhauser Effect (A)</p> Signup and view all the answers

<p>Dynamics of proteins</p> Signup and view all the answers

<p>250</p> Signup and view all the answers

<p>False (B)</p> Signup and view all the answers

<p>They indicate protons in close spatial proximity.</p> Signup and view all the answers

<p>Larger overlap of signals (D)</p> Signup and view all the answers

<p>Conformational dynamics = Indicates protein flexibility NMR-spectroscopy = Used for smaller proteins Disulfide bonds = Contributes to protein stability Relaxation measurements = Confirm dynamics of proteins</p> Signup and view all the answers

<p>more efficient</p> Signup and view all the answers

<p>True (A)</p> Signup and view all the answers

<p>To ensure structures are consistent with known data (D)</p> Signup and view all the answers

<p>Unambiguous assignment of resonances</p> Signup and view all the answers

<p>The angle of diffraction (B)</p> Signup and view all the answers

<p>False (B)</p> Signup and view all the answers

<p>They enhance scattering and allow for phase calculation by binding to protein side chains.</p> Signup and view all the answers

<p>Fourier</p> Signup and view all the answers

<p>~ 6 Å = Shape of the macromolecule &lt; 3 Å = Polypeptide chain tracing &lt; 1.5 Å = Individual non-hydrogen atoms resolved &lt; 1 Å = Location of hydrogen atoms</p> Signup and view all the answers

<p>Multiwavelength Anomalous Dispersion (MAD) (C)</p> Signup and view all the answers

<p>True (A)</p> Signup and view all the answers

<p>The contribution from the atoms to total scattering varies in amplitude and phase.</p> Signup and view all the answers

<p>resolution</p> Signup and view all the answers

<p>&lt; 1.5 Å (A)</p> Signup and view all the answers

<p>False (B)</p> Signup and view all the answers

<p>It is the physical limit for resolution when using copper Kα X-ray radiation.</p> Signup and view all the answers

<p>MAD</p> Signup and view all the answers

<p>Color (C)</p> Signup and view all the answers

<p>True (A)</p> Signup and view all the answers

A technique used to crystallize proteins by gradually increasing the concentration of a precipitant, like polyethylene glycol, in a drop of protein solution. This leads to protein supersaturation and crystal formation.

A technique used to crystallize proteins by gradually increasing the concentration of a precipitant, like polyethylene glycol, in a drop of protein solution. This leads to protein supersaturation and crystal formation.

The smallest repeating unit in a crystal structure that contains all the information to build the entire crystal. It's like the building block of a crystal.

The smallest portion of a crystal structure that cannot be further subdivided by symmetry operations. It's the fundamental repeating unit in the crystal.

The process of X-rays interacting with a crystal, scattering and forming a diffraction pattern. This pattern can be used to determine the structure of the crystal.

A fundamental law in crystallography that describes the conditions under which a diffracted beam emerges from a crystal. It relates the angle of incidence, wavelength, and spacing between crystal planes.

The phenomenon where X-rays scattered by different atoms in a crystal reinforce each other in specific directions, producing bright spots on a detector.

A method used to collect X-ray diffraction data from a crystal by rotating the crystal while exposing it to an X-ray beam.

A method to visualize the protein structure, showing the path of the polypeptide backbone. Useful for comparing different structures.

A representation emphasizing secondary structure elements of a protein. Uses ribbons to highlight alpha helices and beta sheets.

A model showing all individual atoms of each amino acid, representing their bonds. Useful for highlighting interactions between amino acids.

A model where each non-hydrogen atom is shown as a sphere of its van der Waals radius. Useful for identifying ligand binding pockets.

A representation that depicts the protein surface. It highlights electrostatic properties with color.

A measure of the agreement between the crystallographic model and the experimental X-ray diffraction data. It indicates how well the model explains the observed diffraction.

A global measure of model-to-data agreement, calculated similarly to the R-factor. It uses a subset of reflections not used for model refinement to assess the model's accuracy.

A measure of how much a model deviates from standard geometrical parameters. It indicates the quality of the model.

A technique used to examine non-crystalline structures and conformational states of molecules, providing structural information at various resolutions.

A technique using magnetic fields to study the structure and dynamics of molecules. It is useful for studying biomolecules in solution.

Ill-defined regions in a protein structure can suggest flexibility or insufficient data.

NMR relaxation measurements can be used to confirm or rule out protein dynamics.

Proteins are not static, and they can have significant, functional dynamics.

The fitting procedure used to analyze NMR data can affect the results.

NMR is primarily suitable for determining the structures of relatively small proteins.

NMR signal dispersion increases only slightly with protein size, limiting its usefulness for large proteins.

The overlap of NMR signals makes it challenging to assign resonances in large proteins.

NMR is generally used to determine the 3D structures of proteins smaller than approximately 250 amino acids.

The frequency at which a proton resonates in an NMR spectrum is influenced by its chemical environment.

The 1D-NMR spectrum displays resonances along a single axis, representing the chemical shift of protons. It's like a snapshot of the molecule's proton landscape.

The 2D-NMR spectrum is a more complex representation that shows correlations between different protons in a molecule. It's like a map of proton interactions.

In a 2D-NMR spectrum, a crosspeak indicates an interaction between two protons. It's like a connection on the map showing a specific interaction.

The COSY experiment in 2D-NMR can identify protons that are directly connected to each other through covalent bonds up to three bonds apart.

The TOCSY experiment in 2D-NMR detects all protons within the same amino acid, even if they are not directly connected by covalent bonds.

The NOESY experiment in 2D-NMR detects protons that are close in space regardless of whether they are directly connected by covalent bonds.

The NOE (Nuclear Overhauser Effect) is a phenomenon where the magnetization of one proton can affect the magnetization of another proton if they are close in space.

The NOE intensity is inversely proportional to the sixth power of the distance between protons. This makes NOE a powerful tool for determining distances between protons.

Protein structure calculation involves using NMR data to generate a 3D model of a protein. This process relies on NOE and dihedral angle restraints to guide the structure determination.

A technique used to reduce ambiguities in Nuclear Magnetic Resonance (NMR) data that involves analyzing proteins with labeled isotopes.

A measure of how precisely the structure of a protein is determined by NMR, calculated as the average distance between atoms in different structural models.

The number of restraints used in NMR data to define the structure of a protein. A restraint is a piece of experimental data that limits possible positions of atoms.

A region of the 3D space within a protein structure representing the possible combinations of two backbone angles, phi and psi, for a specific amino acid.

A criterion used to assess the quality of a protein structure determined by NMR, indicating how well the structure agrees with the experimental data.

A type of restraint used in NMR structure determination that reflects the distance between protons in the protein. It helps constrain the possible positions of these protons in the structure.

A rule of thumb for assessing the resolution of an NMR-derived protein structure. Generally, structures with higher numbers of restraints per residue are considered to have higher resolution.

A rule of thumb for comparing the resolution of NMR-derived protein structures to X-ray crystallography structures. Structures with a high number of restraints per residue (≥20) are comparable to X-ray structures with a resolution of 2-2.5 Å.

The relationship between the intensity, wavelength, and phase of a diffracted beam in X-ray crystallography. This information is crucial for determining the positions of atoms in a molecule.

The challenge in X-ray crystallography to determine the phase of each diffracted beam. This is essential for reconstructing the electron density map of the molecule.

A method used to solve the phase problem in X-ray crystallography. It involves soaking a crystal with heavy atoms, which bind to the molecule and diffract X-rays differently. This difference in diffraction patterns provides information about the phases.

An X-ray crystallography method that involves using a synchrotron radiation source and crystals containing specific heavy atoms. By varying the wavelength of the X-rays, the scattering from the heavy atoms can be manipulated to solve the phase problem.

A visual representation of the electron density around atoms in a molecule, generated by a mathematical transformation (Fourier transform) using the diffraction pattern.

The smallest distance between two atoms that can still be distinguished as separate points on an electron density map. Higher resolution allows for more detailed structural features to be observed.

A method used to solve the phase problem in X-ray crystallography. Heavy atoms are introduced to the sample, and their positions are determined by comparing the diffraction patterns of the native and heavy-atom-soaking samples. This information is then used to calculate the phases for the native protein.

The application of heavy atoms in X-ray crystallography to solve the phase problem by introducing specific atoms that scatter X-rays strongly.

The ability to replace one component of a molecule with an isomorphous equivalent without altering the molecule's overall structure. This key principle enables techniques like MIR and MAD.

The difference in scattering power of atoms at different wavelengths, particularly around their absorption edges. This effect is utilized in MAD to solve the phase problem.

A high-energy radiation source capable of producing X-rays with tunable wavelengths. This property makes it essential for techniques like MAD.

A type of amino acid that contains selenium, which is a heavy atom. SeMet is often used in MAD experiments to solve the phase problem.

The process of applying a mathematical formula to transform a diffraction pattern into an electron density map, which visually represents the distribution of electrons around the atoms in a molecule.

The degree of detail visible in an electron density map, reflecting the ability to differentiate closely spaced atoms. Higher resolution indicates that smaller features are resolvable.